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Wine and grape Metabolomics Chapters 3-4

The document discusses analytical chemistry, focusing on chromatography as a technique for separating mixture components based on their distribution between a mobile phase and a stationary phase. It outlines various chromatography methods, such as gas and liquid chromatography, alongside factors affecting column efficiency and resolution. Additionally, the document briefly covers mass spectrometry and its applications in analytical chemistry.

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10/04/2019 1 3. Basic Analytical Chemistry Analytical Chemistry – Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified.
10/04/2019 2 Analytical Chemistry – Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified. Injection port Stationary phase detector Mobile phase Sample (mixture) type Mobile phase Stationary phase Detectors (examples) applicability Gas Chromatography (GC) Gases (H2, N2, Ar) Capillary packed columns FID, MS Volatile organic compounds Liquid Chromatography (LC) Liquid (H2O, MeOH) particles UV, MS Analytical Chemistry – Chromatography
10/04/2019 3 Analytical Chemistry – Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified. Pump back pressure major factor of limitations Analytical Chemistry – Chromatography
10/04/2019 4 Analytical Chemistry – Chromatography Analytical Chemistry – Chromatography Injection quantity
10/04/2019 5 Analytical Chemistry – Chromatography Column use Eddy Diffusion (Multipath) Broadening in Chromatography Analytical Chemistry – Chromatography
10/04/2019 6 Longitudinal Diffusion Broadening Analytical Chemistry – Chromatography Analytical Chemistry – Chromatography
10/04/2019 7 Analytical Chemistry – Chromatography Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time time0 tR Theoretical plate number*, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter *Define column efficiency
10/04/2019 8 Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column • Increase column length (?) Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter Height equivalent of a theoretical plate, H H = L/N L: length of the column www.bioanalysis-zone.com
10/04/2019 9 Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column www.chromacademy.com Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column www.chromacademy.com
10/04/2019 10 Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column Analytical Chemistry – Chromatography Isocratic 100% A 0% B 0% C column 100% A 100% B time
10/04/2019 11 Analytical Chemistry – Chromatography Isocratic 90% A 10% B 0% C column 100% A 100% B time Analytical Chemistry – Chromatography Isocratic gradient A B C column 100% A 100% B time
10/04/2019 12 Analytical Chemistry – Chromatography gradient A B C column 100% A 100% B time Analytical Chemistry – Chromatography gradient A B C column 100% A 100% B time
10/04/2019 13 Analytical Chemistry – Chromatography gradient A B C column 100% A 100% B time Analytical Chemistry – Chromatography multi-step gradient A B C column 100% A 100% B time
10/04/2019 14 Analytical Chemistry – Chromatography Gradient Analytical Chemistry – Chromatography Gradient types
10/04/2019 15 Analytical Chemistry – Chromatography 1:1 extract:H2O Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 % 0 100 V026_QC2_RP_neg 1: TOF MS ES- BPI 3.80e3 V049_QC2_RP_neg 2: TOF MS ES- BPI 2.37e3 0.30 mL/min 30 oC 10 µL inj vol 0.28 mL/min 40 oC 10 µL inj vol* Eluent A: H2O 0.1% formic acid Eluent B: CH3OH 0.1% formic acid*10 µL for wines 13 000 psi 10 500 psi back pressure Analytical Chemistry – Chromatography resolution Rs = WB (tR)B (tR)A- 2 WA 2 + WB (tR)B (tR)A-2 WA+ =
10/04/2019 16 Analytical Chemistry – Chromatography Liquid chromatography column stationary phase choice Polar compounds (sugars, organic acids, amino acids) Medium polar compounds (polyphenols, indoles) • Normal phase • HILIC • Amide • Ion-exchange • Reverse phase • C18 Non polar compounds (lipids) • Reverse phase • C30 • Chiral chromatography • Absorption chromatography • Gel filtration chromatography • … Analytical Chemistry – Detectors
10/04/2019 17 Analytical Chemistry – Detectors nm250 300 350 400 450 500 550 caffeic acid nm250 300 350 400 450 500 550 O OH HO OH OH OH cyanidin UV-VIS or DAD detector Analytical Chemistry – Detectors Mass spectrometer detector (single compound)
10/04/2019 18 Analytical Chemistry – Detectors Mass spectrometer detector (mixture compound) Analytical Chemistry – Mass Spectrometry
10/04/2019 19 Analytical Chemistry – Detectors Nuclear magnetic resonance (NMR) spectroscopy Analytical Chemistry – Mass Spectrometry
10/04/2019 20 Young-Shick Hong (2011) NMR-based metabolomics in wine science Analytical Chemistry – Mass Spectrometry Mass Spectrometry
10/04/2019 21 Analytical Chemistry – Mass Spectrometry Electrospray ionization Analytical Chemistry – Mass Spectrometry + + + + + + ++ + + m/z intensity sample ions mass analyzer detector Single quadrupole MS Scan mode MS
10/04/2019 22 Analytical Chemistry – Mass Spectrometry Detector resolution Analytical Chemistry – Mass Spectrometry O OH OH OH OH OH glucose Formula: C6H12O6 Nominal mass: 180 Monoisototic mass: 180,06342 Average mass: 180,156 element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885
10/04/2019 23 Analytical Chemistry – Mass Spectrometry O OH OH OH OH OH glucose Formula: C6H12O6 Nominal mass: 180 Monoisototic mass: 180,06342 Average mass: 180,156 Analytical Chemistry – Mass Spectrometry exercises
10/04/2019 24 Analytical Chemistry – Mass Spectrometry quercetin Formula: CxHxOx Nominal mass: Monoisototic mass: Average mass: element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885 http://www.chemcalc.org/ O O O O OH OH OH OH ellagic acid Analytical Chemistry – Mass Spectrometry Formula: CxHxOx Nominal mass: Monoisototic mass: Average mass: element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885 http://www.chemcalc.org/ OOH OH OH OH OH OOH OH OH OH OH epicatechin catechin
10/04/2019 25 Analytical Chemistry – Mass Spectrometry (mass accuracy) O + O OH OH O O OH OH OH OH O O m/z error in ppm = (MMTheoretical – MMMeasured) MMTheoretical X 106 Analytical Chemistry – Mass Spectrometry + + + + + + ++ + + m/z intensity sample ions mass analyzer detector Single quadrupole MS Scan mode MS
10/04/2019 26 Analytical Chemistry – Mass Spectrometry + + + + + sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS Full scan mode MS + + + ++ + + + ++ + + + ++ m/z intensity Analytical Chemistry – Mass Spectrometry + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS SIM (Single ion monitoring) mode MS + +
10/04/2019 27 Analytical Chemistry – Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor + + Triple quadrupole MS Product ion scan mode MS Analytical Chemistry – Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor + Triple quadrupole MS MRM (Multiple reaction monitoring) scan mode MS
10/04/2019 28 Analytical Chemistry – Mass Spectrometry m/z intensity analyte ions mass analyzer detector mass analyzer + + + In source fragmentation + + + + + + + + + Analytical Chemistry – Mass Spectrometry Single quadrupole MS m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode [M-H]- = 625.1410[M+H]+ = 627.1556
10/04/2019 29 Analytical Chemistry – Mass Spectrometry Single quadrupole MS m/z intensity 611.2 MM: 611.1607 C27H31O16 ESI+ mode [M]+ = 611.1607 O O + O OH OH OH O O OH OH OH OH OH OH OH OH Analytical Chemistry – Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode
10/04/2019 30 Analytical Chemistry – Mass Spectrometry m/z intensity MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity ESI+ mode ESI- mode 301.0303.0 Analytical Chemistry – Mass Spectrometry m/z intensity MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity ESI+ mode ESI- mode 465.1 463.1301.0303.0
10/04/2019 31 Analytical Chemistry – Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode 465.1 463.1301.0303.0 Triple quadrupole MS Analytical Chemistry – Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode 465.1 463.1301.0303.0
10/04/2019 32 Analytical Chemistry – Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity Q ESI- full scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 Q ESI- full scan mode 301.0 179.0 151.0 301.0 257.0 229.0 Analytical Chemistry – Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- Product ion scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- Product ion scan mode 179.0 151.0 257.0 229.0
10/04/2019 33 Analytical Chemistry – Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- MRM scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- MRM scan mode 151.0229.0 Analytical Chemistry – Mass Spectrometry m/z intensity catechin MM: 290.0790 C15H14O6 m/z intensity Q ESI- full scan mode epicatechin MM: 290.0790 C15H14O6 Q ESI- full scan mode 289.0 203.0 123.0 289.0 OOH OH OH OH OH OOH OH OH OH OH 203.0 123.0
10/04/2019 34 Analytical Chemistry – Mass Spectrometry m/z intensity catechin MM: 290.0790 C15H14O6 m/z intensity QqQ ESI- MRM mode epicatechin MM: 290.0790 C15H14O6 QqQ ESI- MRM mode 203.0 OOH OH OH OH OH OOH OH OH OH OH 203.0 Analytical Chemistry – Mass Spectrometry + + + + + + + + sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS + + + + We apply energy to fragment
10/04/2019 35 Analytical Chemistry – Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS + + + + We apply energy to fragment m/z intensity m/z intensity m/z intensity Analytical Chemistry – Mass Spectrometry Why MRM mode is so famous? • velocity
10/04/2019 36 Analytical Chemistry – Mass Spectrometry Why MRM mode is so famous? • Velocity • Selectivity m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- MRM scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- MRM scan mode 151.0229.0 Analytical Chemistry – Mass Spectrometry Why MRM mode is so famous? • Velocity • Selectivity • Sensitivity
10/04/2019 37 Analytical Chemistry – Mass Spectrometry exercises Analytical Chemistry – Mass Spectrometry O + O OH OH O O OH OH OH OH O O Explain the MS spectrum
10/04/2019 38 Analytical Chemistry – High Resolution Mass Spectrometry QTOF MS Orbitrap Analytical Chemistry – High Resolution Mass Spectrometry
10/04/2019 39 Why use high resolution MS? • Resolution – peak capacity • Reliability – mass accuracy • Selectivity • Sensitivity (in scan mode) Analytical Chemistry – High Resolution Mass Spectrometry Metabolomics: Techniques NMR MS (infusion) LC-MS GC-MS CE-MS Sensitivity * *** **** ***** ***** Theoretical plate - - *** **** ***** Linear range ~2 ~5 ~6 ~6 ~6 Robust ***** ** * ** * Reproducibility ***** * * * * Coverage * ** **** *** *** Velocity * ***** ** * *** User friendly * **** *** **** *** Data processing **** ***** **** * ** Identification ***** * ** **** * File dimension ***** ** * * ** Cost * *** **** ** *** Results * * ***** *** *
10/04/2019 40 Metabolomics: facts  Holistic approach: complementary platforms  Multidisciplinary: chemistry + biology + physics + mathematics + informatics  Untargeted: the metabolites are by definition not pre-defined  Unfeasible validation: hundreds to thousands metabolites, many unknown  Self-awareness: minimum reporting standard / levels of annotation Mass Spectrometry (MS) Direct infusion/Imaging Gas Chromatography (GC) Liquid Chromatography (LC) Capillary Electrophoresis (EC) Nuclear Magnetic Resonance (NMR) NMR: up to 100 metabolites few hundreds metabolites ESI- ESI+ Reverse Phase (RP) Normal Phase (NP) few hundreds metabolites few thousands metabolites GCxGC few hundreds metabolites Derivatisation Targeted versus Untargeted Prefer targeted methods when:  You focus in a targeted group or single metabolite (you know what to measure)  You want/need the absolute concentration (except NMR)  You don’t know very good your instrument (analytical chemistry skills)  You are not familiar with compound annotation  You have a poor knowledge of your sample metabolome  You don’t have plenty of time for data analysis / you want fast results  You want to work alone (biology, organic chemistry, biochemistry, analytical chemistry, bioinformatics, chemo-metrics)
10/04/2019 41 4. Snapshots Statistics Measurement types 1. Nominal: Categorical variables with no meaningful order (e.g. color) 2. Ordinal: Categorical variables with meaningful order (e.g. rating, denomination) 3. Interval or Numerical (e.g. concentration) 1. Absolute concentration (reference standard) 2. Relative concentration (ratio of concentration ) 1. Instrumental variability 2. Technical variability (method) 3. Biological variability
10/04/2019 42 Measurement types 1. Instrumental variability 2. Technical variability (method) 3. Biological variability Let’s study chardonnay Measurement types 1. Instrumental variability 2. Technical variability (method) 3. Biological variability
10/04/2019 43 Measurement types 1. Instrumental variability 2. Technical variability (method) 3. Biological variability time(min) Mass (m/z) Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables
10/04/2019 44 Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables
10/04/2019 45 Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Wines Variables
10/04/2019 46 Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Wines Variables Data analysis types 1. Descriptive statistics Mean: the average of the numbers Median: the middle number when the data is put in order from the least to greatest Mode: the number that occurs most often Range: the difference between the lowest and the highest value
10/04/2019 47 Data analysis types 1. Descriptive statistics Data analysis types 1. Descriptive statistics 2. Univariate statistics a b a b concentrazion
10/04/2019 48 Data analysis types 1. Descriptive statistics 2. Univariate statistics a b a b concentrazion Critical value: if lower than 5% or p-value 0,05 is generally accepted Data analysis types 1. Descriptive statistics 2. Univariate statistics a b a b concentrazion Critical value: if lower than 5% or p-value 0,05 is generally accepted
10/04/2019 49 Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneX Concentrazion Y Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneX Concentrazione Y
10/04/2019 50 Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazionA Concentrazion (X,Y) Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneA Concentrazione (X,Y)
10/04/2019 51 Data analysis types a b PCA (Principle component analysis) PC1 PC2 Data analysis types PCA (Principle component analysis) PC1 PC2
10/04/2019 52 Data analysis types PCA (Principle component analysis) Data analysis types PCA (Principle component analysis) http://setosa.io/ev/principal-component-analysis/
10/04/2019 53 ANOVA (Analysis of variance) A B A B concentrazion CC a b c c c One way ANOVA a b c ANOVA (Analysis of variance) D B A B concentrazion CC a b c c c One way ANOVA a,b b,c b,c Comparison between the means of three or more groups
10/04/2019 54 ANOVA (Analysis of variance) A0 concentrazion C0 a b c c c Two ways ANOVA Comparison between the means of three or more groups where two independent variables are considered Ax Bx concentrazion Cx Time 0 Time X a b c c c Time 0 Time X B0 A0 C0 B0 Ax Bx Cx Data analysis types regression classification clustering
10/04/2019 55 Data analysis types supervisedunsupervised -1200 -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 -600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600 t[1] t[4] Scores Comp[4] vs. Comp[1] colored by Variety Chardonnay Grillo Inzolia Muller Pinot grigio QC Traminer Hotelling’s T2 Ellipse (95%) = (559.1; 1139) R2X[4] = 0.06998 R2X[1] = 0.2906 EZinf o 2 - nomacorc2_1QC (M3: PCA-X) - 2015-01-23 12:22:22 (UTC+1) 3 x Grillo 5 x Pinot gris 1 x Inzolia 1 x Muller Thurgau 1 x Chardonnay 1 x Traminer QC
10/04/2019 56 Normalization time concentration time concentration Internal standard time concentration Internal standard IS normalization Scaling Absoluteconcentration Relativeconcentration 0 10000 100%

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Wine and grape Metabolomics Chapters 3-4

  • 1.
    10/04/2019 1 3. Basic AnalyticalChemistry Analytical Chemistry – Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified.
  • 2.
    10/04/2019 2 Analytical Chemistry –Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified. Injection port Stationary phase detector Mobile phase Sample (mixture) type Mobile phase Stationary phase Detectors (examples) applicability Gas Chromatography (GC) Gases (H2, N2, Ar) Capillary packed columns FID, MS Volatile organic compounds Liquid Chromatography (LC) Liquid (H2O, MeOH) particles UV, MS Analytical Chemistry – Chromatography
  • 3.
    10/04/2019 3 Analytical Chemistry –Chromatography Chromatography is a technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated and quantified. Pump back pressure major factor of limitations Analytical Chemistry – Chromatography
  • 4.
    10/04/2019 4 Analytical Chemistry –Chromatography Analytical Chemistry – Chromatography Injection quantity
  • 5.
    10/04/2019 5 Analytical Chemistry –Chromatography Column use Eddy Diffusion (Multipath) Broadening in Chromatography Analytical Chemistry – Chromatography
  • 6.
    10/04/2019 6 Longitudinal Diffusion Broadening AnalyticalChemistry – Chromatography Analytical Chemistry – Chromatography
  • 7.
    10/04/2019 7 Analytical Chemistry –Chromatography Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time time0 tR Theoretical plate number*, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter *Define column efficiency
  • 8.
    10/04/2019 8 Analytical Chemistry –Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column • Increase column length (?) Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter Height equivalent of a theoretical plate, H H = L/N L: length of the column www.bioanalysis-zone.com
  • 9.
    10/04/2019 9 Analytical Chemistry –Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column www.chromacademy.com Analytical Chemistry – Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column www.chromacademy.com
  • 10.
    10/04/2019 10 Analytical Chemistry –Chromatography • Height • Area • Half height • Wh: peak width at half height • Wb: baseline width • tR: retention time Theoretical plate number, N N = 16 tR Wb 2 Column dimension  Particle size  length  Internal diameter How to improve column efficiency Height equivalent of a theoretical plate, H H = L/N L: length of the column Analytical Chemistry – Chromatography Isocratic 100% A 0% B 0% C column 100% A 100% B time
  • 11.
    10/04/2019 11 Analytical Chemistry –Chromatography Isocratic 90% A 10% B 0% C column 100% A 100% B time Analytical Chemistry – Chromatography Isocratic gradient A B C column 100% A 100% B time
  • 12.
    10/04/2019 12 Analytical Chemistry –Chromatography gradient A B C column 100% A 100% B time Analytical Chemistry – Chromatography gradient A B C column 100% A 100% B time
  • 13.
    10/04/2019 13 Analytical Chemistry –Chromatography gradient A B C column 100% A 100% B time Analytical Chemistry – Chromatography multi-step gradient A B C column 100% A 100% B time
  • 14.
    10/04/2019 14 Analytical Chemistry –Chromatography Gradient Analytical Chemistry – Chromatography Gradient types
  • 15.
    10/04/2019 15 Analytical Chemistry –Chromatography 1:1 extract:H2O Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 % 0 100 V026_QC2_RP_neg 1: TOF MS ES- BPI 3.80e3 V049_QC2_RP_neg 2: TOF MS ES- BPI 2.37e3 0.30 mL/min 30 oC 10 µL inj vol 0.28 mL/min 40 oC 10 µL inj vol* Eluent A: H2O 0.1% formic acid Eluent B: CH3OH 0.1% formic acid*10 µL for wines 13 000 psi 10 500 psi back pressure Analytical Chemistry – Chromatography resolution Rs = WB (tR)B (tR)A- 2 WA 2 + WB (tR)B (tR)A-2 WA+ =
  • 16.
    10/04/2019 16 Analytical Chemistry –Chromatography Liquid chromatography column stationary phase choice Polar compounds (sugars, organic acids, amino acids) Medium polar compounds (polyphenols, indoles) • Normal phase • HILIC • Amide • Ion-exchange • Reverse phase • C18 Non polar compounds (lipids) • Reverse phase • C30 • Chiral chromatography • Absorption chromatography • Gel filtration chromatography • … Analytical Chemistry – Detectors
  • 17.
    10/04/2019 17 Analytical Chemistry –Detectors nm250 300 350 400 450 500 550 caffeic acid nm250 300 350 400 450 500 550 O OH HO OH OH OH cyanidin UV-VIS or DAD detector Analytical Chemistry – Detectors Mass spectrometer detector (single compound)
  • 18.
    10/04/2019 18 Analytical Chemistry –Detectors Mass spectrometer detector (mixture compound) Analytical Chemistry – Mass Spectrometry
  • 19.
    10/04/2019 19 Analytical Chemistry –Detectors Nuclear magnetic resonance (NMR) spectroscopy Analytical Chemistry – Mass Spectrometry
  • 20.
    10/04/2019 20 Young-Shick Hong (2011)NMR-based metabolomics in wine science Analytical Chemistry – Mass Spectrometry Mass Spectrometry
  • 21.
    10/04/2019 21 Analytical Chemistry –Mass Spectrometry Electrospray ionization Analytical Chemistry – Mass Spectrometry + + + + + + ++ + + m/z intensity sample ions mass analyzer detector Single quadrupole MS Scan mode MS
  • 22.
    10/04/2019 22 Analytical Chemistry –Mass Spectrometry Detector resolution Analytical Chemistry – Mass Spectrometry O OH OH OH OH OH glucose Formula: C6H12O6 Nominal mass: 180 Monoisototic mass: 180,06342 Average mass: 180,156 element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885
  • 23.
    10/04/2019 23 Analytical Chemistry –Mass Spectrometry O OH OH OH OH OH glucose Formula: C6H12O6 Nominal mass: 180 Monoisototic mass: 180,06342 Average mass: 180,156 Analytical Chemistry – Mass Spectrometry exercises
  • 24.
    10/04/2019 24 Analytical Chemistry –Mass Spectrometry quercetin Formula: CxHxOx Nominal mass: Monoisototic mass: Average mass: element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885 http://www.chemcalc.org/ O O O O OH OH OH OH ellagic acid Analytical Chemistry – Mass Spectrometry Formula: CxHxOx Nominal mass: Monoisototic mass: Average mass: element Isotope Atomic mass (u) % isotopic composition carbon 12C 12,00000 98,93 oxygen 16O 15,99491 99,757 hydrogen 1H 1,00783 99,9885 http://www.chemcalc.org/ OOH OH OH OH OH OOH OH OH OH OH epicatechin catechin
  • 25.
    10/04/2019 25 Analytical Chemistry –Mass Spectrometry (mass accuracy) O + O OH OH O O OH OH OH OH O O m/z error in ppm = (MMTheoretical – MMMeasured) MMTheoretical X 106 Analytical Chemistry – Mass Spectrometry + + + + + + ++ + + m/z intensity sample ions mass analyzer detector Single quadrupole MS Scan mode MS
  • 26.
    10/04/2019 26 Analytical Chemistry –Mass Spectrometry + + + + + sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS Full scan mode MS + + + ++ + + + ++ + + + ++ m/z intensity Analytical Chemistry – Mass Spectrometry + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS SIM (Single ion monitoring) mode MS + +
  • 27.
    10/04/2019 27 Analytical Chemistry –Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor + + Triple quadrupole MS Product ion scan mode MS Analytical Chemistry – Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor + Triple quadrupole MS MRM (Multiple reaction monitoring) scan mode MS
  • 28.
    10/04/2019 28 Analytical Chemistry –Mass Spectrometry m/z intensity analyte ions mass analyzer detector mass analyzer + + + In source fragmentation + + + + + + + + + Analytical Chemistry – Mass Spectrometry Single quadrupole MS m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode [M-H]- = 625.1410[M+H]+ = 627.1556
  • 29.
    10/04/2019 29 Analytical Chemistry –Mass Spectrometry Single quadrupole MS m/z intensity 611.2 MM: 611.1607 C27H31O16 ESI+ mode [M]+ = 611.1607 O O + O OH OH OH O O OH OH OH OH OH OH OH OH Analytical Chemistry – Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode
  • 30.
    10/04/2019 30 Analytical Chemistry –Mass Spectrometry m/z intensity MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity ESI+ mode ESI- mode 301.0303.0 Analytical Chemistry – Mass Spectrometry m/z intensity MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity ESI+ mode ESI- mode 465.1 463.1301.0303.0
  • 31.
    10/04/2019 31 Analytical Chemistry –Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode 465.1 463.1301.0303.0 Triple quadrupole MS Analytical Chemistry – Mass Spectrometry m/z intensity 627.2 MM: 626.1483 C27H30O17 O O O OH OH OH O O OH OH OH OH OH OH OH OH O m/z intensity 625.1 ESI+ mode ESI- mode 465.1 463.1301.0303.0
  • 32.
    10/04/2019 32 Analytical Chemistry –Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity Q ESI- full scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 Q ESI- full scan mode 301.0 179.0 151.0 301.0 257.0 229.0 Analytical Chemistry – Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- Product ion scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- Product ion scan mode 179.0 151.0 257.0 229.0
  • 33.
    10/04/2019 33 Analytical Chemistry –Mass Spectrometry m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- MRM scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- MRM scan mode 151.0229.0 Analytical Chemistry – Mass Spectrometry m/z intensity catechin MM: 290.0790 C15H14O6 m/z intensity Q ESI- full scan mode epicatechin MM: 290.0790 C15H14O6 Q ESI- full scan mode 289.0 203.0 123.0 289.0 OOH OH OH OH OH OOH OH OH OH OH 203.0 123.0
  • 34.
    10/04/2019 34 Analytical Chemistry –Mass Spectrometry m/z intensity catechin MM: 290.0790 C15H14O6 m/z intensity QqQ ESI- MRM mode epicatechin MM: 290.0790 C15H14O6 QqQ ESI- MRM mode 203.0 OOH OH OH OH OH OOH OH OH OH OH 203.0 Analytical Chemistry – Mass Spectrometry + + + + + + + + sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS + + + + We apply energy to fragment
  • 35.
    10/04/2019 35 Analytical Chemistry –Mass Spectrometry + + + + + + + + m/z intensity sample ions mass analyzer detector mass analyzerfragmentor Triple quadrupole MS + + + + We apply energy to fragment m/z intensity m/z intensity m/z intensity Analytical Chemistry – Mass Spectrometry Why MRM mode is so famous? • velocity
  • 36.
    10/04/2019 36 Analytical Chemistry –Mass Spectrometry Why MRM mode is so famous? • Velocity • Selectivity m/z intensity quercetin MM: 302.0427 C15H10O7 m/z intensity QqQ ESI- MRM scan mode O O O O OH OH OH OH ellagic acid MM: 302.0062 C14H6O8 QqQ ESI- MRM scan mode 151.0229.0 Analytical Chemistry – Mass Spectrometry Why MRM mode is so famous? • Velocity • Selectivity • Sensitivity
  • 37.
    10/04/2019 37 Analytical Chemistry –Mass Spectrometry exercises Analytical Chemistry – Mass Spectrometry O + O OH OH O O OH OH OH OH O O Explain the MS spectrum
  • 38.
    10/04/2019 38 Analytical Chemistry –High Resolution Mass Spectrometry QTOF MS Orbitrap Analytical Chemistry – High Resolution Mass Spectrometry
  • 39.
    10/04/2019 39 Why use highresolution MS? • Resolution – peak capacity • Reliability – mass accuracy • Selectivity • Sensitivity (in scan mode) Analytical Chemistry – High Resolution Mass Spectrometry Metabolomics: Techniques NMR MS (infusion) LC-MS GC-MS CE-MS Sensitivity * *** **** ***** ***** Theoretical plate - - *** **** ***** Linear range ~2 ~5 ~6 ~6 ~6 Robust ***** ** * ** * Reproducibility ***** * * * * Coverage * ** **** *** *** Velocity * ***** ** * *** User friendly * **** *** **** *** Data processing **** ***** **** * ** Identification ***** * ** **** * File dimension ***** ** * * ** Cost * *** **** ** *** Results * * ***** *** *
  • 40.
    10/04/2019 40 Metabolomics: facts  Holisticapproach: complementary platforms  Multidisciplinary: chemistry + biology + physics + mathematics + informatics  Untargeted: the metabolites are by definition not pre-defined  Unfeasible validation: hundreds to thousands metabolites, many unknown  Self-awareness: minimum reporting standard / levels of annotation Mass Spectrometry (MS) Direct infusion/Imaging Gas Chromatography (GC) Liquid Chromatography (LC) Capillary Electrophoresis (EC) Nuclear Magnetic Resonance (NMR) NMR: up to 100 metabolites few hundreds metabolites ESI- ESI+ Reverse Phase (RP) Normal Phase (NP) few hundreds metabolites few thousands metabolites GCxGC few hundreds metabolites Derivatisation Targeted versus Untargeted Prefer targeted methods when:  You focus in a targeted group or single metabolite (you know what to measure)  You want/need the absolute concentration (except NMR)  You don’t know very good your instrument (analytical chemistry skills)  You are not familiar with compound annotation  You have a poor knowledge of your sample metabolome  You don’t have plenty of time for data analysis / you want fast results  You want to work alone (biology, organic chemistry, biochemistry, analytical chemistry, bioinformatics, chemo-metrics)
  • 41.
    10/04/2019 41 4. Snapshots Statistics Measurementtypes 1. Nominal: Categorical variables with no meaningful order (e.g. color) 2. Ordinal: Categorical variables with meaningful order (e.g. rating, denomination) 3. Interval or Numerical (e.g. concentration) 1. Absolute concentration (reference standard) 2. Relative concentration (ratio of concentration ) 1. Instrumental variability 2. Technical variability (method) 3. Biological variability
  • 42.
    10/04/2019 42 Measurement types 1. Instrumentalvariability 2. Technical variability (method) 3. Biological variability Let’s study chardonnay Measurement types 1. Instrumental variability 2. Technical variability (method) 3. Biological variability
  • 43.
    10/04/2019 43 Measurement types 1. Instrumentalvariability 2. Technical variability (method) 3. Biological variability time(min) Mass (m/z) Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables
  • 44.
    10/04/2019 44 Data types 1. Asingle sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables
  • 45.
    10/04/2019 45 Data types 1. Asingle sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Data types 1. A single sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Wines Variables
  • 46.
    10/04/2019 46 Data types 1. Asingle sample with several variables measured 2. Two samples or group of samples with one variable measured 3. Two samples or group of samples with several variables measured 4. Three or more samples or group of samples with one variable measured 5. Three or more samples or group of samples with several variables measured Wines Variables Wines Variables Wines Variables Data analysis types 1. Descriptive statistics Mean: the average of the numbers Median: the middle number when the data is put in order from the least to greatest Mode: the number that occurs most often Range: the difference between the lowest and the highest value
  • 47.
    10/04/2019 47 Data analysis types 1.Descriptive statistics Data analysis types 1. Descriptive statistics 2. Univariate statistics a b a b concentrazion
  • 48.
    10/04/2019 48 Data analysis types 1.Descriptive statistics 2. Univariate statistics a b a b concentrazion Critical value: if lower than 5% or p-value 0,05 is generally accepted Data analysis types 1. Descriptive statistics 2. Univariate statistics a b a b concentrazion Critical value: if lower than 5% or p-value 0,05 is generally accepted
  • 49.
    10/04/2019 49 Data analysis types 1.Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneX Concentrazion Y Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneX Concentrazione Y
  • 50.
    10/04/2019 50 Data analysis types 1.Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazionA Concentrazion (X,Y) Data analysis types 1. Descriptive statistics 2. Univariate statistics 3. Multivariate statistics a b a b a b ConcentrazioneA Concentrazione (X,Y)
  • 51.
    10/04/2019 51 Data analysis types a b PCA(Principle component analysis) PC1 PC2 Data analysis types PCA (Principle component analysis) PC1 PC2
  • 52.
    10/04/2019 52 Data analysis typesPCA (Principle component analysis) Data analysis types PCA (Principle component analysis) http://setosa.io/ev/principal-component-analysis/
  • 53.
    10/04/2019 53 ANOVA (Analysis ofvariance) A B A B concentrazion CC a b c c c One way ANOVA a b c ANOVA (Analysis of variance) D B A B concentrazion CC a b c c c One way ANOVA a,b b,c b,c Comparison between the means of three or more groups
  • 54.
    10/04/2019 54 ANOVA (Analysis ofvariance) A0 concentrazion C0 a b c c c Two ways ANOVA Comparison between the means of three or more groups where two independent variables are considered Ax Bx concentrazion Cx Time 0 Time X a b c c c Time 0 Time X B0 A0 C0 B0 Ax Bx Cx Data analysis types regression classification clustering
  • 55.
    10/04/2019 55 Data analysis types supervisedunsupervised -1200 -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 -600-500 -400 -300 -200 -100 0 100 200 300 400 500 600 t[1] t[4] Scores Comp[4] vs. Comp[1] colored by Variety Chardonnay Grillo Inzolia Muller Pinot grigio QC Traminer Hotelling’s T2 Ellipse (95%) = (559.1; 1139) R2X[4] = 0.06998 R2X[1] = 0.2906 EZinf o 2 - nomacorc2_1QC (M3: PCA-X) - 2015-01-23 12:22:22 (UTC+1) 3 x Grillo 5 x Pinot gris 1 x Inzolia 1 x Muller Thurgau 1 x Chardonnay 1 x Traminer QC
  • 56.